Single-molecule spectroscopy studies of several nucleoprotein complexes demonstrated that HIV-1 reverse transcriptase (RT) assumes different orientations, depending on the nature of the nucleic acid substrate. An unexpected outcome was our observation that nonnucleoside RT inhibitors (NNRTIs) induce enzyme binding to the polypurine tract (PPT) in an orientation incompatible with initiating DNA synthesis. Subsequent studies examined tRNA-primed initiation of (-) strand DNA synthesis, showing that the base-paired U5-IR stem immediately downstream of the primer binding site controls enzyme orientation and that this barrier to initiation can be alleviated by including the HIV-1 nucleocapsid (NC) protein. We are continuing single-molecule spectroscopy to examine the effect of NC-containing Gag precursors and RT mutations on (a) tRNA-primed initiation on the HIV-1 genome, (b) tRNALys,3-primed initiation on related lentiviral RNAs, (c) dynamics of termination at the central termination sequence (CTS), and (d) conformational dynamics of a gammaretroviral RT. These projects are combined with new strategies for site-specific placement of fluorophores within RT subunits containing AZ-Phe via Click chemistry.